TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

Abstract

Here, we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. We reveal that this enzyme is TTRAP, a member of the Mg 2+ /Mn 2+ -dependent family of phosphodiesterases, and show that cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase II-induced DNA double-strand breaks. TTRAP is the first human 5'-tyrosine phosphodiesterase to be identified and we suggest this enzyme additionally be denoted tyrosyl DNA phosphodiesterase-2 (TDP2).In an attempt to identify novel human tyrosyl DNA phosphodiesterase activities, we exploited the hypersensitivity of Saccharomyces cerevisiae tdp1Δ rad1Δ double mutant cells to camptothecin (CPT), a topoisomerase I (Top1) poison that induces single-strand breaks (SSBs) with Top1 covalently linked to the 3'-terminus 3, 10 . This strain lacks not only Tdp1 but also Rad1-Rad10 nuclease, which in yeast provides an alternative (endonucleolytic) pathway for removing Top1 from 3'-termini 11,12 . We transformed this strain with a human cDNA library 3 and screened the resulting population of transformants for cellular resistance to CPT. Of six tdp1Δ rad1Δ transformants displaying wild-type levels of CPT resistance, three harboured cDNA clones encoding TDP1 and three harboured cDNA clones encoding TTRAP (TRAF and TNF receptor-associated protein), a protein of unknown function and a putative member of the Mg 2+ /Mn 2+ -dependent phosphodiesterase super-family, with the DNA repair protein AP endonuclease-1 (APE1) being its closest relative 13,14 (Fig.1a and Supplementary Fig.1).The TDP1 and TTRAP cDNA clones recovered in the genetic screen suppressed the CPT sensitivity of tdp1Δ rad1Δ cells to a similar extent (Fig. 1b & data not shown). Whilst the pACT-TTRAP clones encoded TTRAP protein that lacked eight (pACT-TTRAP-2, pACT-TTRAP-3) or twenty-two (pACT-TTRAP-1) residues from the amino-terminus (data not shown), full-length TTRAP similarly suppressed the CPT sensitivity of tdp1Δ rad1Δ (Fig. 1c, left). In contrast, human APE1 protein failed to suppress this sensitivity, suggesting that ability to complement CPT sensitivity in tdp1Δ rad1Δ cells is not a generic feature of metaldependent phosphodiesterases (Fig. 1c, left). Conversely, whereas human APE1 suppressed the sensitivity of AP endonuclease-defective apn1Δapn2Δ tpp1Δ yeast cells to methyl methanesulphonate (MMS)-induced DNA base damage, human TTRAP did not, suggesting that the impact of TTRAP in these experiments was restricted to topoisomerase-mediated DNA damage ( Fig. 1c, right). TTRAP contains four highly conserved motifs that putatively assign this protein to the metal-dependent phosphodiesterase superfamily (see Fig.1a and Supplementary Fig.1). We thus examined whether mutation of two predicted catalytic residues (Fig.1a; E152 and D262) within two of these motifs impacted on the complementation of CPT sensitivity by TTRAP. Indeed, in contrast to wild-type TTRAP protein, ne...

ZATT (ZNF451)–mediated resolution of topoisomerase 2 DNA-protein cross-links

“…In this context, TDP2 modulates cellular (7, 10) and organismal (12) survival following TOP2 targeting anticancer drug treatments, and TDP2 inhibitors hold promise for chemotherapy (13, 14). A critical question in TOP2 biology is how TDP2 accesses the TOP2-DNA phosphotyrosyl chemical bond which is protected within the TOP2 protein shell (15, 16) (Fig.…”

TDP2, TOP2, and SUMO: what is ZATT about?

“…The importance of this enzyme is illustrated by its proposed role in oncogenic translocations, resistance to chemotherapeutic topoisomerase 'poisons' and by causative mutations in the TDP2 gene in Spinocerebellar Ataxia Autosomal Recessive 23 (SCAR23) [4,5]. The denaturation and/or degradation of TOP2cc is thought to be a prerequisite for TDP2 activity, to enable access of the 5′-phosphotyrosyl bond within the active site of topoisomerase [6].…”

Topoisomerase-mediated chromosomal break repair: an emerging player in many games

“…TDP2 activity may reduce chromosomal translocations in prostate tissue by ensuring rapid and accurate resolution of TOP2-mediated PDBs. Consistent with this, depletion of TDP2 in prostate cancer cell lines reduced transcriptional induction of androgen-responsive genes as a result of abortive TOP2 activity 132 . The DNA termini left after TDP2-mediated PDB repair are readily ligatable (FIG.…”